The embryonic neuronal tube is at the outset composed of an undifferentiated neuroepithelium over its entire length. The cells then differentiate, depending on their positions on the anterior/posterior, dorsal/ventral and medium/lateral axes thereby generating various neuronal structures. This differentiation is in part controlled by genetic factors that subdivide the neuronal tube into several histogenic regions. It is now known that numerous transcriptional factors and, more particularly, products encoded by the so-called bHLH “Basic Helix Loop Helix” genes (Caudy et al., 1988, Cell, 55, 1061–67) are involved in this differentiation phenomenon. The proteins of this family have highly conserved amino acid units and more particularly a basic domain followed by Helix 1 and Helix 2. The latter two units are separated by a loop of variable size. In vertebrates, certain products of these bHLH genes participate in the various stages of neurogenesis during neuronal determination and differentiation. By way of representatives of these proteins, there may be mentioned in particular the Math-1 (Akazawa et al., 1995, J. Bol. Chem., 279,8730–38), Mash-1 (Johnson et al., 1990, 346, 858–61), and NeuroD (Lee et al., 1995, Sciences, 268, 836–44) proteins. Evidently, other proteins comprising this bHLH domain are involved in neuronal development at the level of other sites and at variable times. The identification of new bHLH-type proteins would be particularly valuable for increasing our understanding of neurogenesis and therefore be advantageous from the therapeutic point of view.
Members of the basic helix loop helix (bHLH) transcription factor family play a key role in a wide variety of cell fate determination and specification processes. These processes are as diverse as cardial muscle development1, skeletal development2, mesodermal cell determination3, dermal cell differentiation4 and more particularly, myogenesis5 and neurogenesis6.
Our understanding of the molecular basis of vertebrate nervous system development has benefitted from the identification of bHLH factors analogous to those regulating neurogenesis in Drosophila. In the fly, proneural bHLH genes, such as the four genes of the achaete-scute complex (AS-C) and the atonal gene are required for the fate determination of neuronal precursors within the ectoderm6, 7. The expression of proneural genes in Drosophila is restricted to the neural precursor cells by lateral inhibition mediated by the Notch signaling pathway8, 9. The search for vertebrate homologs of the Drosophila proneural genes has led to the identification of numerous genes encoding bHLH proteins. The AS-C homolog Mash1, the atonal homolog Math1 and the most recently identified genes of the neurogenin (ngn) family, including ngn1/Math4C, ngn2/Math4A and ngn3/Math4B/Relax have temporal and spatial expression patterns consistent with a function in neuronal determination10. The three neurogenin genes encode basic helix loop helix transcription factors. Functional analysis of these murine genes has shown that they play a key role in the determination of neuronal cell fate. In contrast, several other genes of the NeuroD family, including NeuroD/Beta2, Math2/Nex1, Math3 are more likely to be involved in differentiation than determination10. Functional analysis of these vertebrate genes has clearly suggested that the overall mechanism controlling neurogenesis is conserved throughout evolution at the fate determination and specification levels. The precise role in the developing nervous system of the putative neuronal determination genes has been investigated by gain-of-function and loss-of-function studies. The overexpression in Xenopus or Zebrafish embryos of some of the latter genes converts non-neural ectoderm into neurons suggesting that these genes encode neuronal determination factors11-14. Analysis of null mutations of Mash1, ngn1 and ngn2 has demonstrated that these genes have true determination functions affecting the development of at least a subset of neuronal cells15-17.